Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), increasing both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomides therapeutic effect are still being defined. We hypothesize that thalidomide induces the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and &beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin induced by SCF is through a PKC-alpha-dependent Ras/Raf/Erk1/2 signaling pathway involving activation of the transcription factor NF-Ya and inhibition of the repressor Coup-TFII. Specific inhibition of PKC-alpha with Go6976 blocked both activation of Erk1/2 and p38 MAPK induced by SCF, and abrogated the SCF increased gamma-globin gene expression. Activation of Erk1/2 plays a critical role in SCF modulated down-stream transcriptional regulators, involving regulation of gamma-globin gene induction. SCF induced nuclear translocation of NF-Ya is required to activate Erk1/2 increased phosphorylation of endogenous Nrf2, which involves up-regulation of thioredoxin, and down-regulation of Coup-TFII. Inhibition of either PKC-alpha or Erk1/2 prevented SCF induced recruitment of NF-Ya, RNA polymerase II and displacement of Coup-TFII repressor from gamma-globin-promoter, indicating that the PKC-alpha-Erk1/2 MAPK pathway contributes to SCF induced the gamma-globin gene induction in adult erythropoiesis. Furthermore, consistent with this concept, SCF induced the gamma-globin gene induction attenuated by inhibition of PKC-alpha or Erk1/2 MAPK. Our data suggest that SCF stimulates the PKC/ROS/Erk1/2 MAPK signaling pathway, which regulates the downstream repressor COUP-TFII (by inhibiting serine/threonine phosphatase 2A activity) and the transcriptional activator NF-Ya (by inducing Ref-1), and that decreased COUP-TFII expression and increased NF-Ya expression induce gamma-globin reactivation in adult erythropoiesis. These observations provide insight into the molecular pathways that regulate gamma-globin augmentation during stress erythropoiesis.